Visualization of avian eggs

ABSTRACT

The invention includes methods of visualizing and manipulating avian eggs employing TPLSM.

RELATED APPLICATION INFORMATION

This application is a continuation-in-part of U.S. patent applicationSer. No. 09/654,293, filed Sep. 1, 2000, the disclosure of which isincorporated in its entirety herein by reference.

BACKGROUND OF THE INVENTION

The avian reproductive system, including that of the chicken, is welldescribed. The production of an avian egg begins with formation of alarge yolk in the ovary of the hen upon which an unfertilized oocyte orovum is positioned. After ovulation or release from the ovary, the yolkand ovum pass into the infundibulum of the oviduct, where the ovum isfertilized if sperm are present, and then moves into the magnum of theoviduct which is lined with tubular gland cells. The tubular gland cellssecrete egg-white proteins, including ovalbumin, lysozyme, ovomucoid,conalbumin and ovomucin, into the lumen of the magnum which is depositedonto the ovum and yolk.

The hen oviduct offers outstanding potential as a protein bioreactorbecause of high levels of protein production and ease of recovery. As aresult, efforts have been made to manipulate avian eggs in order toproduce transgenic chickens expressing exogenous proteins in theoviduct. For example, microinjection of exogenous nucleic acid sequenceshas been performed in order to produce transgenic avians which produceheterologous proteins in the magnum which are subsequently packaged intoeggs laid by the avian. Successful manipulations of avian eggs can belimited by factors including the inability to clearly visualize the eggby standard magnification procedures (e.g., using a light microscope).

Therefore, it is an object of this invention to provide improved methodsfor the visualization of avian eggs and their nuclear structures. It isalso an object of this invention to provide an improved method forvisualization of the nuclear structures in a recipient cell tofacilitate the process of enucleation and subsequent nuclear transfer.It is also an object of this invention to provide an improved method forablation of the nucleus in a recipient cell to facilitate subsequentnuclear transfer.

SUMMARY OF THE INVENTION

In one aspect, the invention is directed to methods for visualizing anavian egg which include visualizing the egg by Two-Photon Laser ScanningMicroscopy (TPLSM). The methods also include visualizing the nucleus ofthe avian egg by TPLSM. The avian egg can be unfertilized (e.g., oocyte)or fertilized (e.g., embryo or zygote). In the case of an embryo, theembryo may be, for example, a stage I, stage II, stage III, stage IV,stage V, stage VI, stage VII, stage VIII, stage IX, stage X, stage XI orstage XII embryo. In one useful embodiment, the embryo is an early stageembryo such as a stage I, stage II or stage III embryo. In oneembodiment, the embryo contains more than one cell. For example, theembryo may contain between 1 and 100,000 cells.

Methods of the invention are also directed to visualizing an avian eggin order to facilitate manipulation of the egg. The inventioncontemplates any useful manipulation of avian eggs in conjunction withviewing the egg by TPLSM. For example, manipulation of the egg mayinclude injection a substance, such as nucleic acid, into the egg and/orinto a nucleus of the egg. In one particular embodiment, the methodsprovide for injection of an artificial chromosome into the avian egg,for example, injection of an artificial chromosome into the nucleus ofthe egg. Nucleic acid injected into the egg typically comprises atransgene. Ovum transfer or ex ovo culture may be used for production ofa transgenic avian after the introduction, e.g., injection, of nucleicacid into an avian egg as disclosed herein.

The avian egg which is visualized and or manipulated as disclosed hereincan be the egg of, for example, and without limitation, a chicken,turkey, duck, goose, quail, pheasant, parrot, finche, hawk, crow,ratite, ostrich, emu or cassowary.

The present invention contemplates cloned cells, cell lines, embryos,and animals and methods for their production, employing two-photonvisualization, ablation or both. Cloned and transgenic avians, includingknock-outs and knock-ins, are also contemplated.

In one embodiment of the invention, two-photon laser scanning microscopy(TPLSM) is used to visualize nuclear structures in a recipient cell. Inone embodiment, following visualization, the cell can be enucleated byany useful method, for example, by two-photon laser-mediated ablation,to provide a recipient cytoplast. It is also contemplated that therecipient cell can be enucleated by cell splitting, aspiration of itsnuclear structure(s), irradiation, or other enucleating procedure. Therecipient cell may be removed from an animal, the nucleus visualized andablated by two-photon laser-mediated ablation. It is contemplated that adonor nucleus can then be inserted into the recipient cell by cellfusion, microinjection, or other useful renucleation procedures. Thereplacement of the recipient cell's nucleus with the donor cell'snucleus yields a reconstructed zygote. It is contemplated that thereconstructed zygote may be activated and allowed to develop to term invivo or in vitro.

Another aspect of the present invention contemplates methods ofproducing a cloned avian comprising nuclear transfer in combination withovum transfer. TPLSM and two-photon laser-mediated ablation is used toperform nuclear transfer wherein the donor nucleus may be a normalkaryotype or may be genetically modified. Accordingly, the replacementof the recipient cell's nucleus with the donor cell's nucleus results ina reconstructed zygote. The ovum may be cultured by ovum transfer,wherein the ovum containing the reconstructed zygote is transferred to arecipient avian or may be cultured in vitro. Once transferred, theembryo develops inside the recipient hen and travels through the oviductof the hen where it is encapsulated by natural egg white proteins and anatural egg shell. The egg which contains endogenous yolk and areconstructed embryo, is laid and can then be incubated and hatched toproduce a chick. The resulting chick may be genetically modified. In oneembodiment, the genetically modified cloned avian carries a transgene inall or some of its cells. After maturation, the transgenic avian may layeggs that contain one or more exogenous protein(s). The combination ofnuclear transfer and ovum transfer allows for the preparation of acloned avian. In another embodiment, ex ovo culture is used instead ofovum transfer to produce the cloned avian.

Another aspect of the present invention contemplates methods ofproducing a transgenic avian by, for example, preparing a transgenicavian, carrying a gene encoding an exogenous protein, using nucleartransfer by two-photon visualization and/or ablation, and allowing theimmature transgenic avian to grow to maturity, wherein the exogenousprotein is secreted into the oviduct lumen of the mature avian anddeposited into eggs laid by the avian. Preferably, the exogenous DNAcomprises a stable transgene and the transgenic avians may be bred andpropagated. In one embodiment, transgenic avians possess the ability tolay eggs that contain one or more desired, exogenous protein(s).

Yet, another aspect of the present invention contemplates methods ofproducing a knock-out or knock-in avian by (i) preparing a knock-out orknock-in avian according to nuclear transfer by two-photon visualizationand/or ablation, and (ii) allowing the immature knock-out or knock-inegg-laying animal to grow to maturity. The knock-out avians are able tolay eggs that contain less than all endogenous proteins normally presentin the egg. This allows for the elimination of potential undesiredsubstances found in the egg (e.g., allergens) or suppression of aspecific agronomic trait. The knock-in sequence may replace all or partof an endogenous gene of the animal by a functional homologous gene orgene segment of another animal.

Cloned non-human cells, cell lines, embryos, and animals, optionallygenetically modified, are encompassed by the instant invention.Transgenic, knock-out, and knock-in animals are also provided. In oneembodiment, reconstituted avian embryos, particularly chick embryos,prepared by transferring the nucleus of a donor cell into a suitablerecipient cell, are provided. The donor cell may be quiescent ornon-quiescent.

Intact avian eggs containing protein(s) exogenous to naturally occurringavian eggs are included in the present invention.

Any useful combination of features described herein is included withinthe scope of the present invention provided that the features includedin any such combination are not mutually inconsistent as will beapparent from the context, this specification, and the knowledge of oneof ordinary skill in the art.

Additional objects and aspects of the present invention will become moreapparent upon review of the detailed description set forth below.

Definitions

The following definitions are set forth to illustrate and define themeaning and scope of the certain terms used to describe the inventionherein.

The term “TPLSM” refers to two-photon laser scanning microscopy. TPLSMrelies upon the phenomenon of two-photon excited fluorescence in whichtwo photons collide simultaneously with a fluorescent molecule. Theircombined energy is absorbed by the fluorophore inducing a fluorescentemission, which is detected by a photomultiplier and converted into adigital image. The major advantage of TPLSM lies in its ability togenerate images of living and optically dense structures for prolongedperiods of time, while not affecting their viability. This is the casebecause TPLSM utilizes biologically innocuous pulsed infrared light thatis able to penetrate much deeper into scattering specimens. Hence thismethod provides the capability for producing noninvasive,three-dimensional, real-time images of the optically dense oocyte (e.g.,avian egg). In addition to visualization, TPLSM may also be used forenucleation.

The terms “ovum” and “oocyte” are used interchangeably herein. Althoughonly one ovum matures at a time, an animal is born with a finite numberof ova. During fertilization, sperm penetrate the blastodisc or germinaldisc, which contains the nuclear material. When sperm enters thegerminal disc an embryo begins to form.

“Egg” or “avian egg” refers to an unfertilized ovum or a fertilizedovum. In particular, “egg” can refer to the germinal disc, which may befertilized or unfertilized, present on the yolk.

A “donor cell” is used herein to describe the source of the nuclearstructure that is transplanted to the recipient cytoplast. All cells ofnormal karyotype, including embryonic, fetal, and adult somatic cells,preferably in a quiescent state, may be nuclear donors. The use ofnon-quiescent cells as nuclear donors has been described as well byCibelli, et al. Science 280:1256-8, 1998, the disclosure of which isincorporated in its entirety herein by reference.

A “recipient cell” is used herein to describe the enucleated recipientcell, for example, an enucleated metaphase I or II oocyte or anenucleated unactivated oocyte, or an enucleated preactivated oocyte.Enucleation may be accomplished by splitting the cell into halves,aspirating the metaphase plate, pronulceus or pronuclei, or even byirradiation. In one embodiment, enucleation is done through two-photonlaser-mediated ablation. Alternatively, TPLSM could be used to guidemechanical enucleation.

A “nucleic acid sequence” or “polynucleotide” includes, but is notlimited to, eucaryotic mRNA, cDNA, genomic DNA, and synthetic DNA andRNA sequences, comprising the natural nucleotide bases adenine, guanine,cytosine, thymidine, and uracil. The term also encompasses sequenceshaving one or more modified nucleotide(s). The terms “polynucleotide”,“oligonucleotide”, and “nucleic acid sequence” are used interchangeablyherein and include, but are not limited to, coding sequences(polynucleotide(s) or nucleic acid sequence(s) which are transcribed andtranslated into polypeptide in vitro or in vivo when placed under thecontrol of appropriate regulatory or control sequences); controlsequences (e.g., translational start and stop codons, promotersequences, ribosome binding sites, polyadenylation signals,transcription factor binding sites, transcription termination sequences,upstream and downstream regulatory domains, enhancers, silencers, andthe like); and regulatory sequences (DNA sequences to which atranscription factor(s) binds and alters the activity of a gene'spromoter either positively (induction) or negatively (repression)). Nolimitation as to length or to synthetic origin are suggested by theterms described herein.

The terms “endogenous nucleic acid sequence” and “endogenous DNA” areused interchangeably herein. The term “endogenous” as it relates tonucleic acid sequences such as coding sequences, control sequences, andregulatory sequences denotes sequences that are normally associated witha particular cell or tissue. Hence, endogenous sequences are found innature. Endogenous proteins are the expression products of endogenousDNA, such as endogenous coding sequences.

The terms “exogenous nucleic acid sequence” and “exogenous DNA” are usedinterchangeably herein. The term “exogenous” as it relates to nucleicacid sequences such as coding sequences, control sequences, andregulatory sequences denotes sequences that are “not” normallyassociated with a particular cell or tissue. Thus, an “exogenous” regionof a nucleic acid is an identifiable segment of nucleic acid within orattached to another nucleic acid molecule that is not found inassociation with the other molecule in nature. Exogenous DNA may beintegrated into the genome of the donor cell or may exist independentlyof the genome of the donor cell. For example, exogenous DNA may not beintegrated into the genome of the donor cell but may exist as part of anon-integrated vector in the donor cell.

The term “transgene” refers to exogenous polynucleotide sequence(s)which can include a desired coding sequence and control sequences inoperable linkage, so that cells transformed with these sequences arecapable of producing the encoded product. In order to effecttransformation, the transgene may be included on a vector. For example,the vector may be an integrative vector which has or can becomeintegrated into the host chromosome. A “transgenic animal” is an animalthat contains one or more exogenous nucleotide sequences in its genome.

The term “knock-out animal” refers to an animal that lacks all or aportion of a specific gene that is normally present in its genome.

The term “knock-in animal” refers to an animal that carries a specificnucleic acid sequence such as a “knock-in sequence” in a predeterminedcoding or noncoding region, wherein the knock-in sequence may beintroduced through methods of recombination, such as homologousrecombination. The recombination event comprises replacing all or partof a gene of the animal by a functional homologous gene or gene segmentof another animal, where the respective knock-in sequence is placed inthe genomic sequence.

“Vector” means a nucleotide sequence useful to introduce DNA into acell. In one embodiment, the vector is comprised of DNA or RNA which maybe single strand, double strand, circular and/or supercoiled. In oneembodiment, the vector contains a coding sequence and a regulatorysequence. The positioning and orientation of the coding sequence withrespect to the regulatory sequence may be such that the coding sequenceis transcribed being at least partially under the “control” of theregulatory sequence.

A “plasmid” is a small, circular DNA vector capable of independentreplication within a bacterial or yeast host cell.

The terms “transformation”, “transduction”, and “transfection” alldenote the introduction of a polynucleotide into a cell, such as anembryonic or somatic cell.

The term “exogenous protein” means a protein or polypeptide notnaturally present in a particular composition, tissue or cell, a proteinthat is the expression product of an exogenous gene, an exogenousexpression construct or a transgene, or a protein not naturally presentin a given quantity in a particular tissue or cell.

The term “avian” means a bird of any known species or type. The termincludes the various know strains of Gallus gallus, or chickens, (forexample, White Leghorn, Brown Leghorn, Barred-Rock, Sussex, NewHampshire, Rhode Island, Ausstralorp, Minorca, Amrox, California Gray,Italian Partridge-colored), as well as turkeys, pheasants, quails, duck,ostriches and other poultry commonly bred for commercial purposes.

DETAILED DESCRIPTION OF THE INVENTION

The invention is drawn to the use of TPLSM in visualizing avian eggs andthe nuclei of avian eggs. For example, the invention includesvisualizing fertilized or unfertilized avian ovum (e.g., fertilized orunfertilized germinal discs) and their nuclear structures. TPLSM isparticularly useful for visualizing these structures because, forexample, the opaque yolk of an egg can substantially obscurevisualization using techniques such as standard light microscopy.

TPLSM is based on two-photon excited fluorescence in which two photonscollide simultaneously with a fluorescent molecule. Their combinedenergy is absorbed by the fluorophore, inducing fluorescent emission,which is detected by a photomultiplier tube and converted into a digitalimage. See Squirrell et al., Nat. Biotechnol. 17:763-7, 1999 and Pistonet al., Trends Cell Biol., 9:66-9, 1999, the disclosure of which isincorporated in its entirety herein by reference. TPLSM allows for thegeneration of images of living, optically dense structures for prolongedperiods of time, while not necessarily affecting their viability. Thisis possible because TPLSM can use biologically innocuous pulsed nearinfrared light, usually at a wavelength of about 700 nm to about 1000nm, which is able to penetrate much deeper into scattering specimens.TPLSM may employ different lasers, such as a mode-locked laser, wherethe wavelength is fixed, or a tunable laser that can be tuned betweenabout 700 nm and about 1000 nm., depending upon the range of emission ofthe dye used. For DAPI and Hoescht 33342 dyes, 720-770 nm is preferred.New fluorophores are being produced with different ranges of emissionand the invention is not limited to the presently available dyes andtheir respective emission ranges.

Lasers used in TPLSM can be grouped into femtosecond and picosecondlasers. These lasers are distinguished by their pulse duration.Femtosecond lasers can be particularly suitable for visualizationwithout harming the specimen, however, the invention is not limitedthereto.

In one embodiment, TPLSM is useful for producing noninvasive,three-dimensional, real-time images of the optically dense avian egg. Inone embodiment, the albumen capsule is removed and the ovum placed in adish with the germinal disc facing the top. Any remnants of the albumencapsule can be removed from the top of the germinal disc. An aqueoussolution, e.g. phosphate-buffered saline (PBS), can be added to preventdrying of the ovum. In one embodiment, a cloning cylinder is placedaround the germinal disc and DAPI in PBS is added to the cylinder. Inanother embodiment, a DAPI-PBS solution is injected into the germinaldisc by using a glass pipette, after which the dye moves into thenuclear structures. In the case of injecting the dye, removal of thealbumen capsule is not necessary. However, injection of nuclei into thedisk can be facilitated in the absence of the capsule. In oneembodiment, following exposure to DNA-specific dyes, images of theinside of the early avian embryo can be generated through the use ofTPLSM. In certain embodiments, visualization is performed after about 10to 15 minutes after administration of the dye. During visualization, thegerminal disc is placed under the microscope objective and the egg isviewed using relatively low laser powers of about 3-6 milliwatts. Theinvention also contemplates the viewing or visualization of avian eggsusing TPLSM without the use of DNA specific dyes.

The invention contemplates the delivery of any useful substance to anegg such as an avian egg (e.g., delivery to the nucleus of the avianegg) in conjunction with viewing by TPLSM. In a particularly usefulembodiment, the invention provides for the delivery of an aqueoussolution to the egg and/or the nucleus, by injection. In one embodiment,the aqueous solution includes a biomolecule such as nucleic acid (e.g.,DNA or RNA). Any useful type of nucleic acid may be employed in thepresent invention. For example, the nucleic acid may be linear orcircular (e.g., open circular or closed circular). In one embodiment,the nucleic acid is associated with protein, for example, a chromosome(e.g., an artificial chromosome) may be delivered to an avian egg ornucleus. The invention also contemplates the delivery of a nucleus tothe egg. In one embodiment, “delivery” means introducing into, forexample, inside of the egg (e.g., into the nucleus of the egg) byinjection.

In one embodiment, the present invention is useful to create atransgenic (e.g., transchromosomic) avian by injecting a nucleic acidcomponent (e.g., an artificial chromosome, see, for example, U.S. patentapplication Ser. No. 11/068,115, filed Feb. 28, 2005, the disclosure ofwhich is incorporated in its entirety herein by reference) into an avianreproductive cell such as a cell of a germinal disc which is atop a yolkand is viewed by TPLSM. In one embodiment, the invention provides for aminimally invasive delivery of DNA or other substance to a germinal discthereby providing for a germinal disc which remains viable afterinjection. In one particularly useful embodiment, an artificialchromosome is delivered into the nucleus of the avian egg.

To produce a transgenic avian, a fertilized ova (stage I embryo) can beisolated from a euthanized hen (female bird), for example, 45 min to 4 hafter oviposition of the previous egg. Alternatively, the eggs can beisolated from hens whose oviducts have been fistulated according to thetechniques of Gilbert & Wood-Gush, J. Reprod. Fertil., 5: 451-453 (1963)and Pancer et al, Br. Poult. Sci., 30: 953-7 (1989), each incorporatedby reference herein in their entireties. In one embodiment, the yolkhaving the fertilized ova is placed in a dish with the germinal discupwards. Ringer's buffer medium can be added to the dish to preventdrying. In one embodiment, nucleic acid is injected into the germinaldisc by visualizing the germinal disc by TPLSM and guiding the injectionneedle of the device into the germinal disc. In one embodiment, a piezounit operably linked to the injection needle can be activated for aperiod of time sufficient for the needle to penetrate the oolemma and ornuclear membrane following which the nucleic acid is injected, e.g.,into the nucleus. Certain aspects of the delivery of nucleic acid intothe avian egg by microinjection are disclosed in U.S. patent applicationSer. No. 11/159,973, filed Jun. 23, 2005, the disclosure of which isincorporated in its entirety herein by reference.

Injected embryos can be surgically transferred to a recipient hen asdescribed, for example, in Olsen & Neher, J. Exp. Zool., 109: 355-66(1948) and Tanaka et al, J. Reprod. Fertil., 100: 447-449 (1994), thedisclosure of which is incorporated herein in its entirety by reference.In one embodiment, the injected embryos are surgically transferred torecipient hens by the ovum transfer method of Christmann et al inPCT/US01/26723, published Aug. 27, 2001, the disclosure of which isincorporated herein by reference in its entirety, and hard shell eggsare incubated and hatched. The embryo is allowed to proceed through thenatural in vivo cycle of albumin deposition and hard-shell formation.The transgenic embryo is then laid as a hard-shell egg which isincubated until hatching of the chick.

In accordance with the present invention, the germinal disc may be agerminal disc of any animal which produces a germinal disc, inparticular avians including, but not limited to, chickens, ducks,turkeys, quails, pheasants and ratites.

In one embodiment, the invention is directed to devices useful for thedelivery of an object or a substance such as an isolated cell nucleus, aspermatozoon or a fluid containing biomolecules such as nucleic acid bymicroinjection into an avian oocyte or embryo. The present invention isalso directed to providing methods of microinjecting an isolated cellnucleus, a spermatozoon or a fluid having a nucleic acid therein, intoan avian embryo or embryonic cell. In one useful embodiment, theinvention provides devices and methods useful for delivering a nucleicacid to an avian embryo or avian embryonic cell. For example, theinvention provides for devices and methods useful for delivering anucleic acid to an avian germinal disc. In one useful embodiment, theinvention provides for the delivery of one or more chromosomes to a germcell or an embryo, for example, delivery into the nucleus of a germinaldisc.

In one useful embodiment, the injection needle can be positioned (i.e.,moved to a certain location) by employing a micromanipulator operablyattached to the needle. The invention contemplates the movement of theneedle provided by the micromanipulator to be in one, two or three axes.That is, the needle can be placed at any useful position on the egg andat any useful angle to the egg and can be moved to pierce the membraneof the egg and the nucleus.

In one embodiment, the microinjection device includes a piezo unit.Typically, the piezo unit is operably attached to the needle to impartoscillations to the needle. However, any configuration of the piezo unitwhich can impart oscillations to the needle is included within the scopeof the invention. Typically, the “piezo drill” provides for a tunablefrequency and amplitude which provides for optimization of the piezo'sperformance (e.g., passage of the needle into the egg, i.e., into agerminal disc).

The oscillations of the needle imparted by the piezo may be in anyuseful direction. For example, and without limitation, the oscillationsmay be side to side, back and forth, up and down, in circular, oval,square, rectangular motions or other patterns or combinations thereof.In one useful embodiment, the oscillations are side to side.

In one embodiment, the piezo unit is operably attached to the needlemeaning the piezo unit is able to impart oscillations to the needle. Inone embodiment, the piezo unit is activated during the penetration ofthe oolemma by the needle. For example, the needle may be a piezoelectrically-driven needle, i.e., the needle punctures the surface ofthe egg (e.g., oolemma) in a manner facilitated (e.g., substantiallyfacilitated) by the action of the piezo unit.

The invention contemplates the employment of any useful frequency ofoscillations imparted to the needle by the piezo. In one embodiment, afrequency of greater than 100 Hz is used. The invention contemplates theupper limit for frequency as being limited by the mechanics of thepiezo. For example, and without limitation, a frequency of between about100 Hz and about 100,000 kHz is within the scope of the invention. Inone useful embodiment, the frequency is between about 100 Hz and about100 kHz, for example, about 500 Hz to about 50 kHz. In one embodiment,the frequency is between about 500 Hz and about 10 kHz, for example,about 500 to about 5 kHz. In one certain embodiment, the frequency isabout 3100 Hz.

The invention contemplates the employment of any useful amplitude ofoscillations imparted to the needle by the piezo. For example, thetravel distance of the needle is contemplated as being between about0.001 nm and about 100 μm. In one embodiment, the travel distance of theneedle is between about 0.11 nm and about 50 μm. In one embodiment, thetravel distance of the needle is about 1 nm to about 20 μm or about 1 nmto about 10 μm. In one useful embodiment, the travel distance of theneedle is about 0.01 μm to about 20 μm. In one particularly usefulembodiment, the travel distance of the needle is about 0.1 μm to about20 μm, for example, about 1.0 μm to about 10 m (e.g., 5 μm or 7.5 μm).

In one particular embodiment, the piezo unit includes one or more of,for example, all of: a Signal Generator (BK Precision Model # 4011A) setto operate at a frequency of 5 KHz; an Amplifier (Physik InstrumenteGmbH, Amplifier: PI-Polytec E-505 PZT-Power Amplifier, Average power 30W, output voltage −20 to +120 V and optimized for 100V PiezoDrive); anda Piezo actuator (Physik Instrumente GmbH, catalog # P-840.10, 5 μmtravel for latitudinal vibration).

The needle may approach the egg from any useful angle. In one usefulembodiment, the longitudinal axis of the needle is visible when viewingthe egg. That is, the TPLSM viewing is not directly above the needle(i.e., the viewing axis is not parallel to the longitudinal axis of theneedle). However, the invention also contemplates the TPLSM viewingbeing directly above the egg (i.e., the viewing axis being parallel tothe longitudinal axis of the needle). Other aspects of needle anglesuseful in the present invention are disclosed in U.S. patent applicationSer. No. 11/159,973, filed Jun. 23, 2005. In one embodiment of theinvention, the needle is coated with a fluorescent dye which facilitatesvisualization of the needle by TPLSM. In another embodiment, the needleis impregnated with a fluorescent dye which facilitates visualization ofthe needle by TPLSM.

The present invention also contemplates methods for producing clonedanimals by nuclear transfer and by combinations of nuclear transfer andembryo transfer. Nuclear transfer allows the cloning of animal species,wherein individual steps are common to the procedures of embryonic,fetal and adult cell cloning. These steps can include, but are notlimited to, preparation of a chromosome-free recipient cell called acytoplast (which involves chromosome removal often referred to asenucleation); donor cell nucleus (nuclear donor) isolation and transferto the cytoplast to produce a reconstructed embryo; optionalreconstructed embryo culture; and embryo transfer to a synchronized hostanimal.

A novel approach to nuclear transfer in animals, employing two-photonvisualization, is described in the instant invention. In one embodiment,the fertilized or unfertilized egg is removed from an animal andmanipulated in vitro, wherein the nucleus or genetic material of the eggis visualized and removed or ablated and a donor nucleus is inserted. Inone embodiment, the donor nucleus is genetically modified. Two-photonlaser scanning microscopy (TPLSM) is used to visualize the nuclearstructures.

The use of light microscopy to visualize the metaphase plate orpronucleus in avian eggs during nuclear transfer can be hindered by thepresence of the yolk, which makes visualization of these nuclearstructures difficult or impossible. But two-photon imaging withfemtosecond lasers operating in the near infrared allows visualizationof nuclear structures without damaging cellular constituents, despitethe unfavorable optical properties of the egg yolk. Prior tovisualization, specimens may be incubated or injected with DNA-specificdyes such as DAPI (4′,6′-diamidino-2-phenylindole hydrochloride) orHoescht 33342 (bis-benzimide). Typically visualization may be performedafter about 10 to 15 minutes of incubation or about 10 minutes afterinjection. During visualization, the germinal disc can be placed underthe microscope objective and the pronuclear structures searched withinthe disk using relatively low laser powers of about 3-6 milliwatts. Inone embodiment, once the structures are found they may be ablated byusing higher laser power or mechanically removed, guided by TPLSM.

Nuclear transfer typically requires the destruction or enucleation ofthe pronucleus before a nuclear donor can be introduced into the oocyte.In order to enucleate the oocyte to produce a cytoplast donor, it isessential to visualize the pronucleus which resides about 25 μm beneaththe ovum's vitelline membrane within the germinal disc. Microsurgery isone method to accomplish pronuclear removal or enucleation. However,two-photon laser-mediated ablation of nuclear structures provides analternative to microsurgery. Higher laser powers than those used forimaging are used for enucleation, with minimal collateral damage to thecell. As during visualization, the wavelength for ablation generallyranges from about 700 nm to 1000 nm, normally about 750 nm. TPLSM andtwo-photon laser-mediated ablation are more efficient than other certainmethods because they can be less operator dependent and less invasive,which results in higher viability of the recipient cell. Followingvisualization, pronuclear structures may be ablated using higher laserpowers of about 30 to about 70 milliwatts.

In one embodiment, enucleation is followed by renucleation, where acultured somatic cell nucleus (nuclear donor) is injected into theenucleated recipient cytoplast. Renucleation may be performed usingTPLSM and a micromanipulation unit comprising a microinjector and amicromanipulator. Following ablation, the nuclear donor may beintroduced into the germinal disc though guided injection usingepiscopic illumination (i.e., light coming through the objective ontothe sample), for example. The reconstructed zygote may then besurgically transferred to the oviduct of a recipient hen to produce ahard shell egg. Alternatively, the reconstructed embryo may be culturedfor 24 hours and screened for development prior to surgical transfer.The egg can be further incubated to generate a cloned chick, optionallygenetically modified. The cloned chick may carry a transgene in all ormost of its cells. After maturation, the transgenic avian may lay eggsthat contain one or more desired, exogenous protein(s). Alternatively,the resulting chick may be a knock-out animal capable of laying eggsthat contain less than all endogenous proteins normally present in theegg. The cloned chick may also be a knock-in chick expressing analternative phenotype or capable of laying eggs having an exogenousprotein therein. The reconstructed egg may be cultured to term using theex ovo method described by Perry (U.S. Pat. No. 5,011,780, issued Apr.30, 1991).

The replacement of the recipient cell's nucleus with the donor cell'snucleus results in a reconstructed zygote. In one embodiment, thecytoplasmic membrane of the cell used as nuclear donor is disrupted toexpose its nucleus to the ooplasm of the recipient cytoplast. Thenuclear donor may be injected into the germinal disc, where it undergoesreprogramming and becomes the nucleus of the reconstructed embryo.Alternatively, a donor cell may be fused to the recipient cell usingmethods well known in the art, for example, by use of fusion-promotingchemicals, such as polyethylene glycol, inactivated viruses, such asSendai virus, or electrical stimulation.

The methodologies of TPLSM visualization and two-photon laser-mediatedablation described herein, can also be used for selective visualizationand destruction of specific structures within germ and/or somatic cells,for example, as used in nuclear transfer in mammalian species and othervertebrate species. The skilled artisan will be able to readily adaptthe methods established for avians described herein to other types ofanimals including, but not limited to, mammals, fish, reptile(s),amphibian(s), and insect(s).

Another aspect of the present invention contemplates methods ofproducing a cloned animal comprising nuclear transfer in combinationwith ovum transfer. Two-photon visualization and ablation may be used toperform nuclear transfer, as described herein. Accordingly, thereplacement of the recipient cell's nucleus with the donor cell'snucleus results in a reconstructed zygote. In one embodiment, pronuclearstage eggs are used as recipient cytoplasts already activated byfertilization. Alternatively, unactivated metaphase II eggs may serve asrecipient cytoplast and activation may thereafter be induced afterrenucleation. The ovum may be cultured by ovum transfer, wherein theovum containing the reconstructed zygote is transferred to a recipienthen. The ovum is surgically transferred into the oviduct of therecipient hen shortly after oviposition. This is accomplished accordingto normal husbandry procedures oviposition, incubation, and hatching;see Tanaka, et al. J Reprod Fertil 100: 447-449, 1994, the disclosure ofwhich is incorporated herein in its entirety by reference.

In another embodiment, the ovum may be cultured to stage X prior totransfer into a recipient hen. More specifically, reconstructed stage Iembryos are cultured for 24-48 hours to stage X. This allows fordevelopmental screening of the reconstructed embryo prior to surgicaltransfer. Stage I embryos are enclosed within a thick albumen capsule.In this procedure, the albumen capsule is removed, after which thenuclear donor is injected into the germinal disc. Subsequently, thecapsule and germinal disc are recombined by placing the thick capsule incontact with the germinal disc on top of the yolk. Embryos develop tostage X at similar rates as those cultured with their capsules intact.At stage X, the embryo is transferred to the oviduct of a recipient hen.

Once transferred, the embryo develops inside the recipient hen andtravels through the oviduct of the hen where it is encapsulated bynatural egg white proteins and a natural egg shell. The egg whichcontains endogenous yolk and an embryo from another hen, is laid and canthen be incubated and hatched to produce a chick. The resulting chickmay carry a transgene in all or most of its cells. Following maturation,the cloned avian may express a desired phenotype or may be able to layeggs that contain one or more desired, exogenous protein(s).

Ovum transfer can facilitate the production of a genetically modifiedavian. Genetically modified avians encompassed by the instant inventioninclude transgenic avians, for example, transgenic avians produced byinjection into an egg (e.g., injection into a nucleus) as disclosedherein, cloned avians and knock-in and knock-out avians.

In another embodiment, ex ovo culture may be used instead of ovumtransfer to produce genetically modified avians. See, for example, Perry(U.S. Pat. No. 5,011,780, issued Apr. 30, 1991). Avians include, but arenot limited to, chickens, ducks, quails, turkeys, pheasants andostriches. The invention disclosed herein also relates to mammals, fish,reptiles, amphibians, and insects.

The present invention provides for cloned egg-laying animals produced bynuclear transfer and by combinations of nuclear transfer and ovumtransfer as described herein. A novel approach of nuclear transfer,employing two-photon visualization and ablation is used to produce thecloned animals. In addition, the present invention encompasses clonedanimals that are genetically modified including, but not limited to,transgenic, knock-out, and knock-in animals. The instant inventionsatisfies the need for a rapid route to the expression and deposition ofexogenous proteins in eggs. Eggs containing protein(s) exogenous to anegg are also provided by the present invention.

The present invention contemplates methods of producing cloned,transgenic animals through nuclear transfer by two-photon visualizationand ablation, and ovum transfer. Transgenic animals may have theirhereditary properties permanently modified by the introduction ofrecombinant DNA into their germ cells. The combination of zygotereconstruction followed by ovum transfer, as disclosed herein, promisesa more efficient and flexible route to accomplish the cloning of animalsand production of transgenics. One use of this technology is themodification of poultry and livestock genomes to improve agronomictraits. Candidate genes, whose introduction or deletion would enhanceagronomic traits, can be targeted through use of the instant invention.Most importantly, the possibility of cloning avian species or producingtransgenic avians promises tremendous gains for the market place. Thenew technology disclosed herein may be used in selective poultrybreeding, leading to enhanced traits in chickens and their eggs.Further, nuclear transfer techniques developed herein (e.g.,laser-mediated selective ablation of nuclear structures of oocytes) alsohave a wide range of applications in fields such as mammaliantransgenesis, genetics, cell therapies, and transplantation.

One aspect of the present invention provides for a method of producing atransgenic animal, comprising the steps of (i) preparing the transgenicanimal according to nuclear transfer by two-photon visualization andoptionally, laser-mediated ablation, and ovum transfer which containsexogenous DNA in its cells, and (ii) allowing the immature transgenicanimal to grow to maturity. In the case of an avian, an exogenousprotein may be secreted into the oviduct lumen of the mature animal anddeposited into eggs laid by the animal. In one embodiment of the instantinvention, the exogenous DNA comprises a transgene and the resultingtransgenic animals can be bred and propagated. Transgenic aviansproduced by the instant invention also possess the ability to lay eggsthat contain one or more desired, exogenous protein(s).

In one embodiment, it is contemplated that transgenes can be introducedinto the ovum of an animal through nuclear transfer by two-photonvisualization and ablation, wherein the nuclear donor contains a desiredexogenous DNA sequence in its genome. One of ordinary skill in the artwill be able to readily adapt conventional methods to insert the desiredtransgene into the genome of the nuclear donor prior to injection of thenuclear donor into the recipient cytoplast, or prior to fusion of thenuclear donor cell with the recipient cell. For example, a vector thatcontains one or more transgene(s) may be delivered into the nucleardonor cell through the use of a delivery vehicle. The transgene is thentransferred along with the nuclear donor into the recipient ovum.Following zygote reconstruction, the ovum can be transferred into thereproductive tract of a recipient hen. In one embodiment, the ovum istransferred into the infundibulum of the recipient hen. Afterreconstruction, the embryo containing the transgene develops inside therecipient hen and travels through the oviduct of the hen where it isencapsulated by natural egg white proteins and a natural egg shell. Theegg is laid and can be incubated and hatched to produce a transgenicchick. The resulting transgenic chick will carry one or more desiredtransgene(s) in its germ line. Following maturation, the transgenicavian may lay eggs that contain one or more desired, exogenousprotein(s) which can be easily harvested.

In one embodiment of the invention, a nuclear donor cell is transfectedwith a vector construct that contains a transgene. Methods fortransfection of somatic cell nuclei are well known in the art andinclude, by way of example, the use of retroviral vectors,retrotransposons, adenoviruses, adeno-associated viruses, naked DNA,lipid-mediated transfection, electroporation and direct injection intothe nucleus. Such techniques, particularly as applied to avians, aredisclosed in Bosselman (U.S. Pat. No. 5,162,215), Etches (PCTPublication No. WO99/10505), Hodgson (U.S. Pat. No. 6,027,722), Hughes(U.S. Pat. No. 4,997,763), Ivarie (PCT Publication No. WO99/19472),MacArthur (PCT Publication No. WO97/47739), Perry (U.S. Pat. No.5,011,780, issued Apr. 30, 1991), Petitte (U.S. Pat. Nos. 5,340,740 and5,656,749), and Simkiss (PCT Publication No. WO90/11355), thedisclosures of which are incorporated herein in their entirety byreference.

Another aspect of the present invention provides for producing aknock-out or knock-in animal, comprising the steps of (i) preparing theknock-out or knock-in animal according to nuclear transfer by two-photonvisualization and/or ablation, and (ii) allowing the immature knock-outor knock-in animal to grow to maturity.

In one embodiment of the instant invention, the knock-out animal hasbeen manipulated such that an endogenous gene has been removed from thegenome of the donor nucleus. This may be accomplished using describedprotocols for the production of knock-out mice, including thetransformation of the nuclear donors with a targeting vector comprisinggenomic DNA containing the desired modification, flanked by positive(neomycin resistance gene for instance) and/or negative (herpes simplexvirus thymidine kinase) or other applicable selectable marker genes,using a number of described strategies such as the so-called “hit andrun” (Hasty, et al., Nature 350:243-6, 1991 and Valancius and Smithies,Mol. Cell Biol. 11:1402-8, 1991), tag and exchange (Askew, et al., Mol.Cell Biol. 13:4115-24, 1993) and double replacement (Stacey, et al.,Mol. Cell Biol. 14:1009-16, 1994).

The resulting knock-out animal can be bred and propagated. Animalsproduced in this fashion are suitable for research purposes, forexample, to study the effects of specific drugs on the breeding ofpoultry and certain agronomic traits. These knock-out animals alsopossess the ability to lay eggs that contain less than all endogenousproteins normally present in the egg, which allows for the eliminationof potential undesired substances found in the egg (e.g., allergens).

In another embodiment of the instant invention, a knock-in animal ismanipulated such that it carries a specific nucleic acid sequence suchas a “knock-in sequence” in a predetermined coding or noncoding regionof its genome. The knock-in sequence may replace all or part of anendogenous gene of the animal by a functional homologous gene or genesegment of another animal. Knock-in animals can be prepared according toa variation of the standard knock-out method, comprising theintroduction of a foreign gene into the targeting vector, in such a waythat the introduced gene would be under the control of the regulatoryelements that normally control the expression of the endogenous gene.See, for example, Le, et al., Proc. Natl. Acad. Sci. USA 87:4712-6, 1990and McCreath, et al., Nature 405:1066-1069, 2000, the disclosures ofwhich are incorporated in their entirety herein by reference.

Another embodiment of the invention provides methods of producing aprotein derived from cloned or otherwise genetically modified egg-layinganimals, e.g., transgenic avians, produced as disclosed herein. Themethods typically include producing a hard-shell egg that containsexogenous protein and then isolating the exogenous protein from the egg.An intact avian egg containing protein exogenous to an avian egg iscontemplated by the present invention. The transgenic animals of theinstant invention include avians that have a transgene encoding anexogenous protein in their oviducts, wherein the avians secrete intotheir eggs the protein expressed by the transgene. A transgenic avianthat makes a human protein (e.g., human interferon) will recognize thissubstance as its own and will therefore not produce an immune responseagainst it. This makes the egg-laying transgenic avian ideally suitedfor the production of large quantities of human protein. In thisrespect, the avian egg provides an ideal container for the production ofrecombinant proteins because its interior may be sterile and containsantibacterial compounds, and it is easily accessible. As a consequence,the purity of the protein products can be improved and their efficacytested more efficiently. Several proteins that may be produced in thisfashion are contemplated by the present invention. These proteinsinclude, but are not limited to, human growth hormone, interferon,β-casein, α−1 antitrypsin, antithrombin III, collagen, factor VIII,factor IX, factor X, fibrinogen, hyaluronic acid, insulin, lactoferrin,protein C, erythropoietin (EPO), granulocyte colony-stimulating factor(G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF),tissue-type plasminogen activator (tPA), feed additive enzymes,somatotropin, and chymotrypsin. Other proteins contemplated forproduction as disclosed herein are disclosed in, for example, U.S.patent application Ser. No. 11/193,750, filed Jul. 29, 2005, thedisclosure of which is incorporated in its entirety herein by reference.

The disclosures of publications, patents, and patent applicationsreferenced in this application are hereby incorporated by reference intothe present disclosure to more fully describe this invention.

It will be apparent to those skilled in the art that variousmodifications, combinations, additions, deletions and variations can bemade in the present invention without departing from the scope of theinvention. For instance, features illustrated or described as part ofone embodiment can be used in another embodiment to yield a stillfurther embodiment. It is intended that the present invention coverssuch modifications, combinations, additions, deletions and variations ascome within the scope of the appended claims and their equivalents.

The following specific Examples are intended to illustrate the inventionand should not be construed as limiting the scope of the claims.

EXAMPLE 1 Preparation of the Recipient Cytoplast

Incubation

Ova were isolated from euthanized hens between 2 and 4 hours afteroviposition of the previous egg. Alternatively, eggs were isolated fromhens whose oviducts have been fistulated. See, for example, Gilbert andWoodgush, Journal of Reproduction and Fertility 5:451-453, 1963 andPander, et al. Br Poult Sci 30:953-7, 1989, the disclosures of which areincorporated in their entirety herein by reference.

Prior to generating images of the early stage avian embryo, incubationof the DNA specific dye was performed according to the followingprotocol: The albumen capsule was removed and the ovum placed in a dishwith the germinal disc facing the top. Remnants of the albumen capsulewere removed from the top of the germinal disc. Phosphate bufferedsaline was added to the dish to prevent drying of the ovum. A cloningcylinder was placed around the germinal disc and 1.0 μg/ml of DAPI inPBS was added to the cylinder. Visualization was performed afterapproximately 15 minutes of incubation.

Injection

Preparation of the egg was done as described for incubation. Followingremoval of the capsule, 10-50 nanoliters of a 0.1 μg/ml solution of DAPIin PBS was injected into the germinal disc using a glass pipette.Visualization was performed approximately 15 minutes after injection.

Visualization

Following incubation, images of the inside of the avian early embryowere generated through the use of TPLSM. The germinal disc was placedunder the microscope objective, and the pronuclear structures weresearched within the central area of the disk, to a depth of 60 μm usinglow laser power of 3-6 milliwatts at a wavelength of 750 nm. Once thestructures were found they were subsequently ablated.

Nuclear Ablation and Enucleation

Pronuclear structures were subjected to laser-mediated ablation. Inthese experiments, an Olympus 20x/0.5 NA (Numerical Aperture) waterimmersion lens was used. The x and y planes to be ablated were definedwith the two photon software, while the z plane (depth) was just under10 μm for this type of objective. Since the pronuclear structure wasabout 20 μm in diameter, the ablation comprised two steps (2 times 10μm). The focal point was lowered to visualize the remaining of thepronucleus, which was subsequently ablated. The laser power used toablate the pronuclei was between 30 and 70 milliwatts at a wavelength of750 nanometers. For the ablation experiments described above, the imagewas zoomed by a factor of 4 to 5, giving an area compression of 16-25fold. Then the power was increased 10-12 fold for a total intensityincrease of 160-300 fold compared to the visualization intensity of 3-6milliwatts. The ablation intensity (power density) is the functionalparameter, i.e. the power increase of 10-12 fold results in ablationpower of 30-70 milliwatts, but the zoom factor compressed this powerinto an area 16-25× smaller giving a power density increase of 160-300fold.

EXAMPLE 2 Preparation of the Nuclear Donor Cell

Isolation of the Donor Nucleus

Fibroblast cells in cultured were trypsinized (0.25% Trypsin and 1 μMEDTA, Gibco catalog #25200-056), centrifuged twice in PBS containing 5%of Fetal Calf Serum and placed in a 60 mm plastic dish in PBS containing5% of Fetal Calf Serum. Using the microscope/micromanipulation unitdescribed below, under transmission light, the nuclear donors were thenisolated by repeated pipetting of the cells, which disrupted thecytoplasmic membrane and released the nucleus from inside the cell.

EXAMPLE 3 Preparation of the Reconstructed Zygote

Injection

A micromanipulation unit, comprising a IM-16 microinjector and aMM-188NE micromanipulator, both from Nikon/Marishige, were adapted to anupright Nikon Eclipse E800. This microscope was adapted to operate underboth transmission and reflective light conditions. This uniqueconfiguration has allowed us to morphologically examine and prepare(isolate the nuclei, as described above) somatic cells in suspension andto load the injection pipette using dry or water immersion lenses underdiascopic illumination or transmitted light. This was followed by promptlocalization and positioning of the germinal disc under the microscopeand subsequent guided injection of the somatic cells, using dry and longdistance lenses under fiber optic as well as episcopic illumination(light coming from the side and through the objectives onto the samplerespectively).

EXAMPLE 4 Ovum Transfer

At the time of laying, recipient hens are anesthetized by wing veininjection with pentobarbital (0.7 ml of a 68 mg/ml solution). At thistime, the infundibulum is receptive to receiving a donor ovum but hasnot yet ovulated. We have also established that pentobarbital is theanesthetic of choice. Feathers are removed from the abdominal area, andthe area is scrubbed with betadine, and rinsed with 70% ethanol. Thebird is placed in a supine position and a surgical drape is placed overthe bird with the surgical area exposed. An incision is made beginningat the junction of the sternal rib to the breastbone and runningparallel to the breastbone. The length of the incision is approximatelytwo inches. After cutting through the smooth muscle layers and theperitoneum, the infundibulum is located. The infundibulum isexternalized and opened using gloved hands and the donor ovum is gentlyapplied to the open infundibulum. The ovum is allowed to move into theinfundibulum and into the anterior magnum by gravity feed. Theinternalized ovum is placed into the body cavity and the incision closedusing interlocking stitches both for the smooth muscle layer and theskin. The recipient hen is returned to her cage and allowed to recoverwith free access to both feed and water. Recovery time for the bird tobe up, moving and feeding is usually within 45 min. of the operation'send. Eggs laid by the recipient hens are collected the next day, set,and incubated in a Jamesway incubator. They will hatch 21 days later.

Alternatively, a hen whose oviduct is fistulated allows the collectionof eggs for enucleation (Gilbert and Woodgush, Journal of Reproductionand Fertility 5:451-453, 1963) and (Pancer, et al. Br Poult Sci 30:953-7, 1989) as mentioned previously, but also the transfer of thereconstructed embryo to a recipient hen for the production of a hardshell egg (Wentworth, Poultry Science 39:782-784, 1960).

EXAMPLE 5 Production of Transgenic Hens by Microinjection of anOvomucoid Promoter-Bacterial Artificial Chromosome Expression Vector

BAC clones OMC24-IRES-LC and OCM24-IRES-HC are used to producetransgenic chickens by microinjection. A detailed description of theseBACs is disclosed in U.S. patent application Ser. No. 11/047,184, filedJan. 31, 2005, the disclosure of which is incorporated in its entiretyherein by reference. Briefly, each BAC includes a 70 kb chickenovomucoid gene region with a coding sequence for either a heavy chain(HC) or light chain (LC) of a particular human IgG1 antibody. The HC andLC sequences are under the translational control of an internal ribosomeentry site (IRES) which is inserted in the 5′ UTR of the ovomucoid generegion.

The BACs are linearized by enzymatic restriction digest. The digestedDNA is phenol/CHCl₃ extracted, ethanol precipitated, suspended in 0.25 MKCl and is diluted to a working concentration of approximately 60 μg/ml(30 μg/ml OMC24-IRES-LC and 30 μg/ml OMC24-IRES-HC) with SV40 T antigennuclear localization signal peptide (NLS) being added yielding apeptide:DNA molar ratio of 100:1 (Collas and Alestrom, 1996, Mol.Reprod. Develop. 45: 431-438, the disclosure of which is incorporated byreference in its entirety). The DNA samples are allowed to associatewith the SV40 T antigen NLS peptide by incubation at room temperaturefor 15 minutes.

Introduction of the DNA-NLS complex into an avian egg is accomplished bymicroinjection employing a microinjection needle in conjunction withTPLSM. Briefly, TPLSM is used to visualize the germinal disc or zygoteand the tip of the injection needle is inserted into the germinal discsuch that the DNA-NLS complex can be injected directly into a nucleuscontained in the germinal disc.

An injection needle, mounted on a micromanipulator, comprising a drawnout glass capillary tube coated with a thin layer of DAPI is employed toinject the DNA-NLS complex into nuclei contained in avian embryos. StageI White Leghorn chicken embryos are immersed in Ringer's buffer andapproximately 10 to 50 nanoliters of a 0.1 μg/ml solution of DAPI in PBSis injected into each germinal disc. After incubation for approximately15 minutes the nuclei within the germinal disc are visualized usingTPLSM in conjunction with an Olympus 20x/0.5 NA (Numerical Aperture)water immersion lens. Low laser power is used, for example, about 1-6milliwatts at a wavelength of 750 nm. Approximately 10 nanoliters of theDNA-NLS mixture is injected into each of one or more nuclei in theembryos with the positioning of the needle and progress of the injectionbeing monitored by TPLSM.

Injected embryos are surgically transferred to recipient hens by ovumtransfer according to the method of Christmann et al. (see, for example,U.S. patent application Ser. No. 10/679,034, filed Oct. 2, 2003, thedisclosure of which is incorporated herein in its entirety by reference)and hard shell eggs are incubated and hatched. See, Olsen and Neher,1948, J. Exp. Zoo. 109: 355-366, the disclosure of which is incorporatedin its entirety herein by reference.

Genomic DNA samples from one-week old chicks are analyzed for thepresence of OMC24-IRES-LC and OMC24-IRES-HC by PCR. Female chicks thattest positive for the transgene are grown to maturity and lay eggscontaining the antibody, present primarily in the egg white.

EXAMPLE 6 Production of Transchromosomic Chickens Using SatelliteDNA-Based Artificial Chromosomes

Satellite DNA-based artificial chromosomes, as described in Lindenbaumet al Nucleic Acids Res (2004) vol 32 no. 21 el72, the disclosure ofwhich is incorporated in its entirety herein by reference, are isolatedby a dual laser high-speed flow cytometer as described previously. See,for example, de Jong, G, et al. Cytometry 35: 129-133, 1999, thedisclosure of which is incorporated by reference herein in its entirety.

The flow-sorted chromosomes are pelleted by centrifugation of a 750 μlsample containing approximately 10⁵ chromosomes in injection buffer at2500×g for 30 min at 4° C. The supernatant, except the bottom 75microliters (μl) containing the chromosomes, is removed resulting in aconcentration of about 1000 chromosomes per μl of injection buffer. See,Monteith, et al. Methods Mol Biol 240: 227-242, 2004, the disclosure ofwhich is incorporated by reference herein in its entirety.

Embryos for this study are collected from 24-36 week-old hens fromcommercial White Leghorn variety of G. gallus. Embryo donor hens areinseminated weekly using pooled semen from roosters of the same breed toproduce fertile eggs for injection.

On the day of egg collection, fertile hens are euthanized 2 h postoviposition by cervical dislocation. Typically, oviposition is followedby ovulation of the next egg after about 24 minutes (Morris, PoultryScience 52: 423-445, 1973). The recently ovulated and fertilized eggsare collected from the upper magnum region of the oviduct under sterileconditions and placed in a glass well and covered with Ringers' Medium(Tanaka, et al. J Reprod Fertil 100: 447-449, 1994) and maintained at41° C. until microinjection.

Injection of artificial chromosomes into a nucleus of a stage I embryois achieved using TPLSM in combination with microinjection essentiallyas disclosed in example 5. Chromosomes are injected into a nuclearstructure of the stage I embryos. Each injected nucleus is injected withapproximately 1-5 chromosomes.

Following microinjection, the embryos are transferred to the oviduct ofrecipient hens using an optimized ovum transfer (OT) procedure (Olsen, Mand Neher, B. J Exp Zool 109: 355-66, 1948), with the exception that thehens are anesthetized by isofluorane gas. Typically, about 26 h afterOT, the recipient hens lay a hard shell egg containing the manipulatedovum. Eggs are incubated for 21 days in an incubator until hatching ofthe birds. Transchromosomic birds are identified by PCR analysis of DNArecovered from blood samples.

To verify the chromosomes are intact in the transchromosomic avians,metaphase spreads from fibroblast cells derived from founders are madeand analyzed as described previously (Garside and Hillman (1985)Experientia 41: 1183-1184).

All references cited herein are incorporated by reference herein intheir entirety and for all purposes to the same extent as if eachindividual publication, patent or patent application is specifically andindividually indicated to be incorporated by reference in its entiretyfor all purposes.

The citation of any publication is for its disclosure prior to thefiling date and should not be construed as an admission that the presentinvention is not entitled to antedate such publication by virtue ofprior invention.

While this invention has been described with respect to various specificexamples and embodiments, it is to be understood that the invention isnot limited thereto and that it can be variously practiced with thescope of the following claims.

1. A method comprising: visualizing a nucleus of an avian egg by TPLSM.2. The method of claim 1 wherein a substance is injected into thenucleus.
 3. The method of claim 1 wherein the nucleus is injected withnucleic acid.
 4. The method of claim 1 wherein the egg is an oocyte. 5.The method of claim 1 wherein the avian egg is an egg of an avianselected from the group consisting of avians of chicken, turkey, duck,goose, quail, pheasant, parrot, finche, hawk, crow, ratite, ostrich, emuand cassowary.
 6. A method comprising: visualizing a nucleus of an avianembryo by TPLSM.
 7. The method of claim 6 wherein the embryo comprisesmore than one cell.
 8. The method of claim 6 wherein the embryo containsbetween 1 and 100,000 cells.
 9. The method of claim 6 wherein asubstance is injected into the nucleus.
 10. The method of claim 6wherein the nucleus is injected with nucleic acid.
 11. The method ofclaim 10 wherein the nucleic acid comprises a transgene.
 12. The methodof claim 6 wherein the avian egg is an egg of an avian selected from thegroup consisting of avians of chicken, turkey, duck, goose, quail,pheasant, parrot, finche, hawk, crow, ratite, ostrich, emu andcassowary.
 13. The method of claim 6 wherein the egg is an early stageembryo.
 14. The method of claim 6 wherein the egg is selected from thegroup consisting of a stage I, stage II, stage III, stage IV, stage V,stage VI, stage VII, stage VIII, stage IX, stage X, stage XI and stageXII embryo.
 15. A method comprising visualizing an avian egg using TPLSMand injecting an artificial chromosome into a nucleus.
 16. The method ofclaim 15 wherein the egg is an oocyte.
 17. The method of claim 15wherein the egg is a zygote.
 18. The method of claim 15 wherein theavian egg is an egg of an avian selected from the group consisting ofavians of chicken, turkey, duck, goose, quail, pheasant, parrot, finche,hawk, crow, ratite, ostrich, emu and cassowary.
 19. The method of claim15 wherein the embryo is an early stage embryo.
 20. The method of claim15 wherein the embryo is selected from the group consisting of a stageI, stage II, stage III, stage IV, stage V, stage VI, stage VII, stageVIII, stage IX, stage X, stage XI and stage XII embryo.